The 2EF Antibody Targets a Unique N-Terminal Epitope of Trop-2 and Enhances the In Vivo Activity of the Cancer-Selective 2G10 Antibody

Simple Summary We sought recognition of Trop-2 within difficult-to-reach, densely packed tumor sites. The 2EF mAb was developed, and demonstrated efficient access to Trop-2 at cell–cell junctions in breast cancer cells in culture and in prostate tumors that were not accessible to benchmark anti-Trop-2 antibodies. The 2EF antibody was shown to inhibit the growth of cancer cells in vitro, with the highest activity at high cell density. Correspondingly, 2EF showed the highest anticancer activity on densely packed, Trop-2-expressing tumors. The 2EF mAb enhances the activity of the cancer-only binding 2G10 mAb against tumors in vivo, opening novel avenues for Trop-2-targeted therapy. Abstract Trop-2 proteolytic processing in cancer cells exposes epitopes that were specifically targeted by the 2G10 antibody. We sought additional recognition of Trop-2 within difficult-to-reach, densely packed tumor sites. Trop-2 deletion mutants were employed in immunization and screening procedures, and these led to the recognition of a novel epitope in the N-terminal region of Trop-2, by the 2EF antibody. The 2EF mAb was shown to bind Trop-2 at cell–cell junctions in MCF-7 breast cancer cells, and in deeply seated sites in prostate cancer, that were inaccessible to benchmark anti-Trop-2 antibodies. The 2EF antibody was shown to inhibit the growth of HT29 colon tumor cells in vitro, with the highest activity at high cell density. In vivo, 2EF showed anticancer activity against SKOv3 ovarian, Colo205, HT29, HCT116 colon and DU-145 prostate tumors, with the highest impact on densely packed tumor sites, whereby 2EF outcompeted benchmark anti-Trop-2 antibodies. Given the different recognition modes of Trop-2 by 2EF and 2G10, we hypothesized the effective interaction of the two mAb in vivo. The 2EF mAb was indeed demonstrated to enhance the activity of 2G10 against tumor xenotransplants, opening novel avenues for Trop-2-targeted therapy. We humanized 2EF by state-of-the-art CDR grafting/re-modeling, yielding the Hu2EF for therapy of Trop-2-expressing tumors in patients.

This led to the generation of the 2G10 monoclonal antibody (mAb), which selectively recognizes cleaved Trop-2 in transformed cells [20][21][22][23]. However, Trop-2 molecules form dimers and multimers at cell-cell junctions [24][25][26], which may hamper Ab binding in tightly packed tumor cell masses. We thus went on to search for mAb that could efficiently bind Trop-2 in densely arrayed cancer sites. Trop-2 deletion mutants were utilized in immunization and screening procedures, and these led to the isolation of the 2EF mAb, that targets a novel epitope in the Trop-2 N-terminal region.
The 2EF mAb was shown to access Trop-2 at cell-cell junctions in breast MCF-7 cancer cells in culture and at deeply seated sites in prostate cancer, that were inaccessible to benchmark anti-Trop-2 mAb. The 2EF mAb diminished HT29 colon tumor cell growth in vitro, with the highest activity being shown on cells growing at high density. In vivo, 2EF inhibited the growth of Trop-2-expressing SKOv3 ovarian, Colo205, HT29, HCT116 colon and DU-145 prostate xenotransplants. The highest anticancer impact was reached on densely packed, established tumors, rather than on isolated tumor cells, whereby 2EF outcompeted benchmark anti-Trop-2 mAbs. We humanized 2EF by state-of-the-art complementarity-determining regions (CDR) grafting/remodeling, yielding Hu2EF. The 2EF mAb was shown to enhance the activity of the cancer-specific 2G10 [20], opening novel avenues for Trop-2-targeted anticancer therapy.

Generation of the Anti-Trop-2 Murine mAb
Deletion mutants of the Trop-2 extracellular domain were used as immunogens in Balb/c mice and for screening of hybridoma clones by ELISA and flow cytometry [21,22]. Differential binding against Trop-2 deletion mutants allowed for the isolation of the N-terminal regiontargeting 2EF mAb. The anti-Trop-2 AbT16 [27,28] and the irrelevant mAb p181Bg were utilized as Trop-2-binding versus Trop-2-non-binding control mAb, respectively.

Flow Cytometry
Fluorescence analysis and cell sorting were performed, as described [31], on fluorescenceactivated cell analyzers and sorters (FACS Aria III, Canto II, Becton Dickinson, Sunnyvale, CA, USA). mAb were conjugated to Alexa488, 546 or 633 (Life Technologies, Waltham, MA, USA) for direct cell staining. To improve signal-to-noise ratios and the detection of transfectants stained with Alexa488-mAb, subtraction of cell autofluorescence and displacement of Alexa488-stained cells in the red channel were performed essentially as described [32,33].

Immunofluorescence and Confocal Microscopy
Cells grown on glass coverslips were fixed with 4% paraformaldehyde in PBS for 20 min. Permeabilization and blocking were performed in a medium with 10% Fetal Bovine Serum (FBS) and 0.1% saponin. Live cells on glass coverslips were stained in medium with 10% FBS at 37 • C for 5 min and fixed after staining. Slides were analyzed by immunofluorescence (IF) with LSM-510 META and LSM800 (Zeiss, Oberkochen, Germany) confocal microscopes. Three laser beams were used, emitting at wavelengths of 488 nm (argon ion laser, 200 mW, 2-5% applied laser power), 543 nm (diode laser, 1 mW, 50-100% applied laser power), and 633 nm (diode laser, 5 mW, 50% applied laser power). HFT 488/543 or 488/543/633 beam-splitters were used, as needed for multi-color fluorochrome-conjugated Ab analysis, with band-pass emission filters 505 nm to 550 nm (green channel); long-pass 560 nm (for two-color analysis), or band-pass 560 nm to 615 nm (for three-color analyses) in the orange channel; long-pass 650 nm in the deep-red channel. Images were acquired in Multiplex mode, i.e., via sequential acquisition of individual laser lines/fluorescence channels, to prevent cross-channel fluorescence spill-over. Detector gains of ≤770 V were applied to minimize electronic noise. Amplifier gains were ≤2.8. Images were acquired in 1024 × 1024 pixel format, except where indicated. Images were captured as averages of four sequential acquisitions, using 40×/1.2, 63×/1.4 oil DIC objectives (Plan-Apochromat; Zeiss) [20]. Densitometric analysis of independent channel acquisition was performed on representative samples of Trop-2-expressing prostate cancer.

Construction of the Chimeric 2EF IgG1/k Ab
The 2EF VH and VL exons were cloned between the SpeI and HindIII sites (for VH) or the NheI and EcoRI sites (for VL) of a mammalian expression vector carrying human g1 and κ constant regions, to generate a chimeric 2EF IgG1/κ Ab (Ch2EF). Genes encoding 2EF VH and VL were synthesized as individual exons, with splice donor signals at the 3 end of the coding region, that were derived from the mouse germline JH3 and Jκ2 sequences, respectively.

Strategy for 2EF Humanization
The VH sequence of the human M17751 cDNA (M17751 VH) [35] and L02325 cDNA (L02325 VH) [36] were chosen as acceptors for humanization. The human VH CDRs were replaced with the murine 2EF VH CDRs, together with selected framework amino acids that were mutated into their murine counterparts. The amino acid sequences of the resulting humanized VH, Hu2EF VH4 and Hu2EF VH5, respectively, are as in PCT WO201608765.
The Vκ region of the human Z46622 cDNA (Z46622 VL) was chosen as acceptor for humanization. The human VL CDRs were replaced with the murine 2EF VL CDRs, together with selected framework amino acids that were mutated into their murine counterparts. The amino acid sequence of the resulting humanized Hu2EF VL1 is as in PCT WO201608765.
The addition of the R38K mutation to Hu2EF VH4 and VH5 led to the generation of Hu2EF VH7 and VH6, respectively. The VL-M4L and VL-Q100G mutations were combined into the Hu2EF VL1 to generate Hu2EF VL2. The pHu2EF-7 vector that expressed the VH7 and VL2 genes was engineered to carry a puromycin resistance (pHu2EF-7-puro).
Expression vectors carrying Hu2EF-4 and Hu2EF-5 variants were transfected into HEK293 cells for transient expression [31]. Antigen binding of transiently expressed antibodies to Trop-2 was analyzed by ELISA in microtiter plates coated with 0.1 µg/mL of rhTrop2 and by flow cytometry analysis of TROP2 MTE4-14 transfectants.

Experimental Tumors
Tumor cell lines and TROP2 transfectants [29] were injected subcutaneously (5-10 × 10 6 cells) into 8-week-old female athymic CD1-Foxn1 nu/nu mice (Charles River Laboratories, Calco, Lecco, Italy). The tumor longest/shortest diameters (D/d) were measured every 5-7 days. Tumor volumes were calculated as for an ellipsoid (Dxd 2 /2) [10]. Unless indicated, treatment with anti-Trop-2 or irrelevant (p181bg) mAb was performed by weekly intraperitoneal administration of 30 mg/kg of Ab in PBS, for 4 weeks starting from the day of the inoculation or when the tumors reached 100 mm 3 of volume.

Statistical Analysis
One-tailed t test was used for the comparison of matrices of Ab-binding densitometry values in confocal microscopy images. EC 50 values in ADCC activity curves were calculated from dose-response data fitted to a 4-parameter-logistic non-linear regression model. ANOVA [39] and t test implementing a post-hoc Bonferroni correction were used to comparatively assess tumor growth curves. Data were analyzed using Sigma Stat 4.0 (SPSS Science Software UK Ltd., Chicago, IL, USA) and GraphPad Prism 7 (GraphPad Software Inc., La Jolla, CA, USA).

Results
We discovered that activation of Trop-2 as a driver of tumor progression requires proteolytic cleavage by ADAM10 [9,10]. This led to the development of the 2G10 mAb, which selectively recognizes ADAM10-cleaved Trop-2 in transformed cells [21][22][23]. However, Trop-2 molecules form dimers and higher-order multimers at cell-cell contacts [24][25][26]. This may hamper Ab binding in difficult-to-penetrate tumor cell masses. We thus searched for mAb that could efficiently bind Trop-2 at cell-cell junctions in densely arrayed cancer sites.

Generation of mAbs Directed against Accessible Trop-2 Sites in Tightly Packed Tumor Cells
We designed recognition strategies for distinct regions of Trop-2 to generate mAb with selective reactivity against the N-terminal region of Trop-2, i.e., at a distant site from the 2G10 target region and from the Trop-2 immunodominant epitope [19,28,40]. Trop-2 deletion mutants were utilized for hybridoma screening and epitope mapping [21,22], by ELISA, flow cytometry and fluorescence microscopy on cleaved/activated or wild type (wt) recombinant Trop-2. This and the lack of competition versus the 2G10 mAb family showed that the 2EF mAb recognizes a distinct target epitope in the Trop-2 N-terminus.

Effective Binding of 2EF to Tumor Cells at Deeply Seated Cancer Sites
We went on to assess whether 2EF can effectively recognize Trop-2 in difficult-to-reach regions in densely packed tumor cells. IF staining of MTE4-14/Trop-2 transfectants showed efficient recognition of Trop-2 by 2EF ( Figure 1A). We then showed that 2EF gains access to Trop-2 at cell-cell junctions in breast MCF-7, which grow in culture as tightly packed cell islands. Much less efficient binding of Trop-2 at cell junctions was shown by 2G10 and by the benchmark AbT16 mAb ( Figure 1B).
We went on to assess whether 2EF can effectively recognize Trop-2 in difficult-toreach regions in densely packed tumor cells. IF staining of MTE4-14/Trop-2 transfectants showed efficient recognition of Trop-2 by 2EF ( Figure 1A). We then showed that 2EF gains access to Trop-2 at cell-cell junctions in breast MCF-7, which grow in culture as tightly packed cell islands. Much less efficient binding of Trop-2 at cell junctions was shown by 2G10 and by the benchmark AbT16 mAb ( Figure 1B). We then assessed whether 2EF could gain access to Trop-2 at deeply seated tumor sites, that were inaccessible to benchmark anti-Trop-2 mAb. Multiplex confocal microscopy IF analysis of prostate cancer revealed deeper penetration/effective binding of 2EF to cancer cells, as opposed to 2G10 and to the immunodominant epitope-binding AbT16 ( Figure 2).
Comparative 2EF and 2G10 binding were assessed with Fiji/ImageJ 2.9.0. Normalized pixel values were obtained in regions of interest (ROI) of the tumor. A threshold of differential intensity of 2EF versus 2G10 signals was identified as the divergence point of binding curves versus depth of penetration in tumor islands (Table S1). In other words, we determined the threshold distance from the tumor stroma whereby the 2G10 signal started to fall, whereas that of 2EF remained high. This was computed to be 35.6 ± 6.0 µ m (mean ± SEM; range: 10-100 µ m; n = 15). The largest differential intensity/deepest penetration of 2EF versus 2G10 was shown to be 61.6 ± 8.2 µ m (mean ± SEM; range: 21.5-140.0 µ m; p = 0.0102). The ratio of the areas-under-the-curve of 2G10 versus 2EF in ROI was 55 ± 0.03% (mean ± SEM; range: 44.8-73.3%; p = 0.0028), indicating an almost two-fold increase in We then assessed whether 2EF could gain access to Trop-2 at deeply seated tumor sites, that were inaccessible to benchmark anti-Trop-2 mAb. Multiplex confocal microscopy IF analysis of prostate cancer revealed deeper penetration/effective binding of 2EF to cancer cells, as opposed to 2G10 and to the immunodominant epitope-binding AbT16 ( Figure 2).
Comparative 2EF and 2G10 binding were assessed with Fiji/ImageJ 2.9.0. Normalized pixel values were obtained in regions of interest (ROI) of the tumor. A threshold of differential intensity of 2EF versus 2G10 signals was identified as the divergence point of binding curves versus depth of penetration in tumor islands (Table S1). In other words, we determined the threshold distance from the tumor stroma whereby the 2G10 signal started to fall, whereas that of 2EF remained high. This was computed to be 35.6 ± 6.0 µm (mean ± SEM; range: 10-100 µm; n = 15). The largest differential intensity/deepest penetration of 2EF versus 2G10 was shown to be 61.6 ± 8.2 µm (mean ± SEM; range: 21.5-140.0 µm; p = 0.0102). The ratio of the areas-under-the-curve of 2G10 versus 2EF in ROI was 55 ± 0.03% (mean ± SEM; range: 44.8-73.3%; p = 0.0028), indicating an almost two-fold increase in efficiency in penetration/binding of 2EF at central tumor areas. Notably, no Ab penetration/staining was detectable in the lumen of the prostate cancer gland-like structure (Table S1).
Grade 2 polynomial curves were utilized to fit the density sequences of 2G10 and 2EF signals in ROI (Table S1). A comparison of curve parameters (y, x 2 and R 2 ) confirmed the higher absolute values of 2EF binding versus 2G10 at deeply seated sites in prostate cancer. Moreover, in essentially all cases, 2G10 binding curves showed a higher curvature/larger differential binding versus 2EF, which showed a smoother data distribution/higher homogeneity in staining patterns (Table S1). efficiency in penetration/binding of 2EF at central tumor areas. Notably, no Ab penetration/staining was detectable in the lumen of the prostate cancer gland-like structure (Table  S1K). Grade 2 polynomial curves were utilized to fit the density sequences of 2G10 and 2EF signals in ROI (Table S1). A comparison of curve parameters (y, x 2 and R 2 ) confirmed the higher absolute values of 2EF binding versus 2G10 at deeply seated sites in prostate cancer. Moreover, in essentially all cases, 2G10 binding curves showed a higher curvature/larger differential binding versus 2EF, which showed a smoother data distribution/higher homogeneity in staining patterns (Table S1).

Engineering of Chimeric and Humanized 2EF
To prevent eliciting a human anti-mouse Ab response upon administration to patients [41], the 2EF mAb was humanized. Cloned 2EF VH and VL exons were transferred into a mammalian expression vector carrying human g1 and κ constant regions to generate the chimeric 2EF IgG1/κ Ab (Ch2EF).
The VH sequence encoded by the human M17751 cDNA (M17751 VH) [35] was chosen as an acceptor for CDR grafting. At framework positions 44, 48, 67, 69 and 71, where the three-dimensional model of the 2EF variable regions suggested significant contact with the CDRs, human amino acids were reverted to the corresponding murine residues, to generate the Hu2EF-4 construct. A parallel humanized VH (Hu2EF VH5) was designed using the VH sequence encoded by the human L02325 cDNA (L02325 VH) [36]. At framework positions 48, 67, 69 and 71, human amino acid residues were substituted by the corresponding mouse residues to prevent a steric clash with CDR regions. The three anti-Trop-2 mAb were utilized simultaneously. Individual signals for each fluorophore were acquired independently, in a sequential manner across fluorescence channels. Magnified regions of interest are indicated by white rectangles. White arrowheads indicate the tumor regions with higher differential penetration/binding capacity of 2EF-Alexa488. Scale bars, 50 µm.

Engineering of Chimeric and Humanized 2EF
To prevent eliciting a human anti-mouse Ab response upon administration to patients [41], the 2EF mAb was humanized. Cloned 2EF VH and VL exons were transferred into a mammalian expression vector carrying human g1 and κ constant regions to generate the chimeric 2EF IgG1/κ Ab (Ch2EF).
The VH sequence encoded by the human M17751 cDNA (M17751 VH) [35] was chosen as an acceptor for CDR grafting. At framework positions 44, 48, 67, 69 and 71, where the three-dimensional model of the 2EF variable regions suggested significant contact with the CDRs, human amino acids were reverted to the corresponding murine residues, to generate the Hu2EF-4 construct. A parallel humanized VH (Hu2EF VH5) was designed using the VH sequence encoded by the human L02325 cDNA (L02325 VH) [36]. At framework positions 48, 67, 69 and 71, human amino acid residues were substituted by the corresponding mouse residues to prevent a steric clash with CDR regions.
The human Vκ region encoded by the Z46622 cDNA (Z46622 VL) was chosen as an acceptor for CDR grafting. At position 49, where the three-dimensional model of the 2EF variable regions indicated significant contact with the CDRs, the human amino acid was substituted with the corresponding murine residue.

Ab-Dependent Cell-Mediated Cytotoxicity by Hu2EF
ADCC can mediate a considerable fraction of mAb anticancer activity [44]. The capacity of Hu2EF mAb to mediate ADCC was measured using effector Jurkat cells stably expressing the V158 (high affinity) FcγRIIIa receptor and a firefly luciferase reporter gene, driven by an NFAT response element, which becomes activated when the FcγRIIIa receptor is engaged in ADCC. The target MCF-7 breast cancer cells were selected as expressing endogenous Trop-2 at levels corresponding to average amounts in primary human cancers [8] (Figure 5). Different amounts of purified mAb (range 10 pM-1 µM) were added to monolayers of MCF-7 target cells, together with Jurkat effector cells, at the indicated effector/target cell ratios. Efficient lysis of cancer cells by Hu2EF was observed, with a good EC 50 of 0.3 nM ( Figure 5).

Ab-Dependent Cell-Mediated Cytotoxicity by Hu2EF
ADCC can mediate a considerable fraction of mAb anticancer activity [44]. The capacity of Hu2EF mAb to mediate ADCC was measured using effector Jurkat cells stably expressing the V158 (high affinity) FcγRIIIa receptor and a firefly luciferase reporter gene, driven by an NFAT response element, which becomes activated when the FcγRIIIa receptor is engaged in ADCC. The target MCF-7 breast cancer cells were selected as expressing endogenous Trop-2 at levels corresponding to average amounts in primary human cancers [8] (Figure 5). Different amounts of purified mAb (range 10 pM-1 µ M) were added to monolayers of MCF-7 target cells, together with Jurkat effector cells, at the indicated effector/target cell ratios. Efficient lysis of cancer cells by Hu2EF was observed, with a good EC50 of 0.3 nM ( Figure 5).

Inhibition of Cancer Cell Growth by 2EF
Cell confluency in culture was previously shown to affect Trop-2 proteolysis in transformed cells [9] and activation pathways for cell growth [6]. We thus analyzed the impact of 2EF versus 2G10 on HT29 colon cancer cells growing in culture at high versus low cell density. The 2G10 mAb did inhibit HT29 cell growth better than 2EF at low cell density. However, the 2EF mAb fared as well as 2G10 in high-density cell cultures ( Figure 6A,B).

Inhibition of Cancer Cell Growth by 2EF
Cell confluency in culture was previously shown to affect Trop-2 proteolysis in transformed cells [9] and activation pathways for cell growth [6]. We thus analyzed the impact of 2EF versus 2G10 on HT29 colon cancer cells growing in culture at high versus low cell density. The 2G10 mAb did inhibit HT29 cell growth better than 2EF at low cell density. However, the 2EF mAb fared as well as 2G10 in high-density cell cultures (Figure 6 A,B). These findings supported a model of high capacity of 2EF to recognize Trop-2 in densely arrayed cancer cells. We went on to explore whether 2EF maintained such capacity in vivo. The therapeutic efficacy of the 2EF versus 2G10 against HT29 human cancer xenografts growing in nude mice was assessed. Tumor-bearing mice were treated either These findings supported a model of high capacity of 2EF to recognize Trop-2 in densely arrayed cancer cells. We went on to explore whether 2EF maintained such capacity in vivo. The therapeutic efficacy of the 2EF versus 2G10 against HT29 human cancer xenografts growing in nude mice was assessed. Tumor-bearing mice were treated either at injection or when tumors reached an average volume of 0.1 cm 3 ("individual cell" versus "established tumor" models, respectively). Mice in the control group received an irrelevant isotype-matched mAb or the anti-Trop-2 AR47A6.4.2 mAb [45], as an activity reference. The 2EF mAb showed anticancer activity in both models. In the established cancer model 2EF outcompeted the benchmark anti-Trop-2 mAbs ( Figure 6C,D).

Xenograft Growth Inhibition by 2EF
We then challenged 2EF activity against other Trop-2-expressing tumors. Significant growth inhibition by 2EF was shown against ovarian cancer SKOv-3 (p = 0.0063 by ANOVA test with Bonferroni post-hoc correction versus control mAb), with higher activity than AR47A6.4.2. Corresponding findings were obtained against the colon cancer Colo205 and the metastatic colon cancer HCT-116 U5.5 [9,10] (Figure 7). This demonstrated high anticancer activity of 2EF across tumor models, supporting it as a broadly applicable anticancer reagent. model 2EF outcompeted the benchmark anti-Trop-2 mAbs (Figure 6 C,D).

Xenograft Growth Inhibition by 2EF
We then challenged 2EF activity against other Trop-2-expressing tumors. Significant growth inhibition by 2EF was shown against ovarian cancer SKOv-3 (p = 0.0063 by ANOVA test with Bonferroni post-hoc correction versus control mAb), with higher activity than AR47A6.4.2. Corresponding findings were obtained against the colon cancer Colo205 and the metastatic colon cancer HCT-116 U5.5 [9,10] (Figure 7). This demonstrated high anticancer activity of 2EF across tumor models, supporting it as a broadly applicable anticancer reagent.  The 2EF and 2G10 mAb recognize cancer-expressed Trop-2 in a sharply distinct manner. Hence, we hypothesized that they could effectively interact in cancer cell killing. Athymic nude mice were subcutaneously injected with human DU-145 prostate cancer cells. Injected mice were randomized and treated with 30 mg/kg mAb weekly until sacrifice. Mice treated with 2EF plus 2G10 were administered half dose (15 mg/kg) of each mAb weekly until sacrifice. The AR47A6.4.2 and AbT16 anti-Trop-2-immunodominant site mAb were used as benchmarks. Treatment began when tumors reached an average volume of 0.1 cm 3 . Our findings showed the efficacy of both 2EF and 2G10. Remarkably, though, the highest efficacy was revealed in the combination treatment group (Figure 8), suggesting enhancement of 2G10 anticancer activity by 2EF.
The 2EF and 2G10 mAb recognize cancer-expressed Trop-2 in a sharply distinct manner. Hence, we hypothesized that they could effectively interact in cancer cell killing. Athymic nude mice were subcutaneously injected with human DU-145 prostate cancer cells. Injected mice were randomized and treated with 30 mg/kg mAb weekly until sacrifice. Mice treated with 2EF plus 2G10 were administered half dose (15 mg/kg) of each mAb weekly until sacrifice. The AR47A6.4.2 and AbT16 anti-Trop-2-immunodominant site mAb were used as benchmarks. Treatment began when tumors reached an average volume of 0.1 cm 3 . Our findings showed the efficacy of both 2EF and 2G10. Remarkably, though, the highest efficacy was revealed in the combination treatment group (Figure 8), suggesting enhancement of 2G10 anticancer activity by 2EF. We did not observe any adverse effect in treated mice versus controls, as indicated by normal behavior, clinical appearance and lack of variation in body weight during treatment. Correspondingly, we did not observe any adverse effect in treated Cynomolgous monkeys as compared with control primates (manuscript in preparation).

Discussion
High-potency Trop-2-targeted therapy for advanced cancer is urgently needed [17]. However, next-generation approaches remain hampered by the expression of Trop-2 in normal tissues [6,[8][9][10]28,46], including the epidermis, endometrium, esophagus, tonsil, lung, kidney, salivary glands and breast [6,21,22]. Such on-target/off-tumor binding was shown to potentially lead to unmanageable toxicity [17,18]. We discovered that activation of Trop-2 as a driver of tumor progression requires proteolytic cleavage by ADAM10 We did not observe any adverse effect in treated mice versus controls, as indicated by normal behavior, clinical appearance and lack of variation in body weight during treatment. Correspondingly, we did not observe any adverse effect in treated Cynomolgous monkeys as compared with control primates (manuscript in preparation).
However, Trop-2 molecules form stable dimers [24] and multimers at cancer cell-cell contacts [25,26]. Hence, target epitopes in Trop-2 may become less accessible to Ab binding in tightly packed cancer cell masses. We thus searched for a mAb with improved access to tightly packed cells in culture and in tumor xenotransplants. Through Trop-2 structurefunction informed analysis [9,24,25] and deletion mutagenesis-based immunization and screening strategies, we succeeded in generating the 2EF mAb, that was shown to recognize a novel epitope in the N-terminal regions of Trop-2.
Confocal microscopy analysis of 2EF binding to Trop-2 in breast MCF-7 cancer cells, which grow in culture as tightly packed cell islands, showed that 2EF can efficiently bind Trop-2 at cell-cell junctions, at variance with the 2G10 and benchmark AbT16 mAb. Multiplex confocal microscopy analysis of prostate cancer samples correspondingly showed much deeper 2EF penetration in cancer cell islands, as opposed to benchmark immunodominant epitope-binding mAb. Consistently, 2EF showed powerful antitumor activity in multiple Trop-2-expressing preclinical tumor models, among them the SKOv3 ovarian, Colo205, HT29, HCT116 U5.5 colon and DU-145 prostate cancers. Remarkably, the highest anticancer impact was reached in the most difficult-to-treat, densely packed established tumors, rather than on isolated tumor cells, whereby 2EF outcompeted all benchmark anti-Trop-2 mAb that were tested.
In order to obtain a mAb that could be repeatedly administered to patients without eliciting a human anti-mouse Ab response [41], the 2EF mAb was humanized by state-ofthe-art CDR grafting/remodeling. Trop-2 binding profiles of Hu2EF-7 versus the Ch2EF, Hu2EF-4, Hu2EF-5 and Hu2EF-6 intermediates, as determined by ELISA and competition flow cytometry, were shown to be similar across distinct cancer cell types, indicating Hu2EF-7 as a successfully humanized form of mouse 2EF.
The effector functions of recombinant therapeutic IgG are dependent on the glycosylation of the Fc region [42]. A strategy to improve binding to the Fc receptor is to modify mAb glycosylation states [47], as low fucose IgG1 exhibit higher in vitro and in vivo ADCC activity compared to highly fucosylated IgG [43]. As both CHO and NS-0 cells possess intrinsic fucosyl-transferase activity, we expressed Hu2EF-7 in YB2/0 cells, a fucosylationlow rat myeloma [37]. Fucosylation-low Hu2EF-7 efficiently bound Trop-2 targets, in a comparable, if not better, manner versus the parental mAb, suggesting this as a viable means for therapeutic approaches in cancer patients.

Conclusions
Recent findings support the cancer-selective Hu2G10 as instrumental for next-generation anti-Trop-2 ADC [21][22][23]. The efficacy of 2EF against several tumor models, its capacity to reach deeply seated cancer sites and its lack of toxicity in animal models support 2EF as a novel candidate and an efficient anti-Trop-2 therapeutic mAb. The efficient humanization of 2EF makes Hu2EF a candidate for the development of novel anti-Trop-2 ADC. The enhancement of the 2G10 cancer-specific mAb in vivo, and the expected reduction in on-target/off-tumor toxicity pave the way for novel approaches for Trop-2-targeted therapy. It will be equally interesting to assess how this innovative concept may work in combination or in sequence versus other anti-Trop-2 mAb with different target epitopes, e.g., SG. Conflicts of Interest: E.G. is an inventor in PCT WO201687651 and WO201784763. M.T. is an inventor in PCT WO201784763. S.A. is an inventor in PCT WO201089782, WO201687651 and WO201784763. S.A. is the founder of Oncoxx Biotech Srl and Mediterranea Theranostic Srl. The sponsors had no role in the design and conduct of this study, nor in the collection, analysis and interpretation of the data, nor in the preparation, review or approval of the manuscript.